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measured by imaging a point source, for example, a small sub-resolution fluorescent

bead (0.1mm), and imaging how the point is spread out in the microscope. Since it is

assumed that the real image of the bead should be a point, it is possible to calculate the

amount of distortion in the image of the bead imposed by the imaging system. The

actual image of the point can then be restored using a mathematical function, which

can be applied to any subsequent images collected under identical settings of the

microscope.

Early versions of the deconvolution method were relatively slow; for example, it

could take some algorithms in the order of hours to compute a single optical section.

Deconvolution is now much faster using today’s fast computers and improved soft-

ware, and the method compares favourably with the confocal approach for producing

optical sections. Deconvolution is practical for multiple-label imaging of both fixed

and living cells, and excels over the scanning methods for imaging relatively dim

and thin specimens, for example yeast cells. The method can also be used to remove add-

itional background from images that were collected with the LSCM, the spinning disk

microscope or a multiple photon microscope.

4.3.5 Total internal reflection microscopy

Another area of active research is in the development of single molecule detection

techniques. For exampletotal internal reflection microscopy (TIRF)uses the proper-

ties of an evanescent wave close to the interface of two media (Fig. 4.16), for example,

the region between the specimen and the glass coverslip. The technique relies on the

fact that the intensity of the evanescent field falls off rapidly so that the excitation of

any fluorophore is confined to a region of just 100 nm above the glass interface. This

is thinner than the optical section thickness achieved using confocal methods and

allows the imaging of single molecules at the interface.

Water

Glass

Vesicles and microtubules

outside evanescent field

Vesicles and microtubules

inside evanescent field

EVANESCENT FIELD

~100 nm

Fig. 4.16Total internal reflection microscopy (TIRF). A 100-nm thick region of excitation is produced at the

glass–water interface when illumination conditions are right for internal reflection. In this example only those

vesicles and microtubules within the evanescent field will contribute to the fluorescence image at 100 nm

Z-resolution.

122 Microscopy